In the United States, approximately 8 to 12 million people are affected with peripheral artery disease (PAD), a form of atherosclerotic disease. PAD can reduce blood flow in the lower limbs, resulting in pain, loss of motility, foot ulcers and the potential for loss of limb. This end result, termed critical limb ischemia (CLI), is treated surgiclly or endoscopically in an effort to enhance blood flow to the limb. Of patients affected by CLI, 20-30% are not suitable for revascularization procedures and may have to be amputated. This has prompted clinical investigation into both medical therapies to achieve therapeutic revascularization as well as cellular therapies. We have described for the first time that an insoluble matrix can control cellular differentiation of mesenchymal stem cells (MSCs) towards vascular cells without additional soluble signals. Our preliminary studies show that MSCs cultured in a hydrogel matrix express genotypic, phenotypic and morphologic characteristics of endothelial cells and pericytes in the absence of additional cytokines. In vivo results indicate that these phenotypic changes lead to increased neovascularization, which may be an enabling step for the functional improvement of a regenerable tissue like skeletal muscle. Therefore, we are interested in understanding the process of vasculogenesis from progenitor cells for the purpose of enabling muscle regeneration. Central to our understanding of this process is the necessity of being able to track stem cells and the resulting newly formed vasculature. An essential requirement for stem cell labels is that they are not toxic over time. It is also essentil that imaging must not alter the behavior or fate of marked stem cell populations. We propose here to utilize spherical gold nanoparticles, plasmonic nantracers, due to their excellent biocompatibility, as well as high and tunable optical absorption properties. Several approaches are available to either track stem cells or to measure neovascularization, but none of the approaches can monitor both simultaneously. Therefore, the overall goal of the current proposal is to quantify blood vessel growth kinetics and function in vivo using nanotracer-enhanced, high-resolution combined ultrasound and photoacoustic (US/PA) imaging. A secondary goal is to be able to quantify revascularization in a model system in which we are able to correlate the extent and structure of blood vessels with quantitative measures of muscle function. The combination of nanotracers with US/PA imaging results in a unique single system approach capable of quantifying blood vessel development from progenitor cells. This will allow us to answer fundamental questions regarding MSC involvement in blood vessel growth as well as validate a clinically translatable solution for tissue regeneration. PUBLIC HEALTH RELEVANCE: Early clinical trials in patients with peripheral arterial disease resulting in critical limb ischemia have demonstrated the safety of autologous stem cell therapies and suggested modest improvements in reperfusion and limb salvage. A major challenge to the development of clinically applied stem cell therapy remains the lack of efficient stem cell tracking methods and in vivo visualization of neovascularization. Therefore, the overall goal of the current proposal is to quantify blood vessel growth kinetics and function in vivo using nanotracer-enhanced, high-resolution combined ultrasound and photoacoustic (US/PA) imaging.